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Mimulus guttatus Lab report

We constructed a double digest restriction associated DNA study on Mimulus guttatus. The first step was collecting samples, which we did on two field trips which can be read about here and here . Next, we extracted DNA from the samples we collected as well as samples collected by Alec detailed here. Next, we double digested our DNA using two restriction enzymes detailed here. These enzymes cut up the genome into many pieces. Next, we ligated unique DNA barcodes onto each of our individuals. The next step was using PCR for two purposes: to add a second unique and to test if our library construction was successful. Our PCR was successful as evidenced by a photo taken by Professor Paul. After the test PCR, we did a larger reaction of 25 micrometers that is identical to the previous one. This is the last step we were able to do as a class. In a perfect world, we would do the following other steps. The next step would be size selection. Size selection selects DNA of specific sizes, specifically, we would target ~400-600 bp. Size selection can be done in three different ways, one is using an automated system called pippin prep of which we have one housed in the Suni lab #Suni. A second way is to use gel extraction. Or, finally, magnetic beads can be used to isolate DNA. After size selection, we would then normalize our DNA samples, meaning bringing all of our DNA samples to approximately the same concentration. Having equal concentrations makes equal numbers of DNA fragments more likely to be sequenced. The final step would be to combine all of our size selected normalized PCR products into one vessel. Then, we would run these samples on any alumina sequencer- our class would run it on our in-house iSeq 1000 (Wall-e). Sequencing would take approximately 16 hours and, if successful, would generate tens of millions of reads. These data would be run through a bioinformatics pipeline. Ultimately, we would align these sequence data with the published Mimulus guttatus genome and call SNPs. Finally, we would use these SNPs to infer population differentiation using a metric like FST and assess population genetic diversity looking at things like the number of alleles, allelic diversity, etc. Based on what I know about Mimulus guttatus, I might expect populations that are genetically divergent. #MolecularEcologyForever

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Lab entry 12

After double digesting and ligating an adapter to the DNA, we did a test PCR. We did this to test for successful library construction of the samples.

  1. First, we made a master mix for the RADSeq using the following:
Master Mix Rxn: 1 Rxns: 11
NEB One-Taq 2x Master Mix 8 uL 88 uL
Forward Primer (10mM) PCR1X .4 uL 4.4 uL
Reverse Primer (10mM) PCR26 .4 uL 4.4 uL
Pure H2O 6.2 uL 68.2 uL
Master Mix Total: 15 ul 165 ul
Library DNA Template 1 ul  
Total reaction volume 16 uL  

 

2. We ran PCR1 on BIORAD #1/2 using 5 uL of the total reaction volume and 2 uL of loading dye.

3. Then, we ran the the products of PCR1 for each sample on 1.5% agarose gel with a 100 bp ladder at 130 V for 40 minutes.

The test PCR had samples #17-24.

For the final PCR, we used the same steps with the following master mix:

Master Mix Rxn: 1 Rxns: 11
Phusion DNA Polymerase .31 uL 3.41 uL
5X Phusion HF buffer 6.25 uL 69 uL
Forward Primer (10mM) PCR1_X 1.56 uL 17.2 uL
Reverse Primer (10mM) PCR2_1 1.56 uL 17.2 uL
DNTPs 10 mM .63 uL 6.93 uL
DMSO .94 uL 10.3 uL
Pure H2O 10.75 uL 118.25 uL
Master Mix Total: 22 ul 242.04 ul
Library DNA Template 3 ul  
Total reaction volume 25 uL  

We used the same samples #17-24. However, for samples 21 and 22 we used PCR27 as the reverse primer instead. This was a mistake made because we ran out of our original master mix and used another groups.

 

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Lab entry 11

Our next step for this lab was to do DD-RADSeq. First, we double digested our DNA samples. To do this, we followed the following steps:

  1. Double digested 100-1000 ng of high quality genomic DNA with selected restriction enzymes, using a digestion buffer appropriate for both enzymes.
  2. Then, I placed 6 uL of each sample’s DNA in the well of a PCR tube, storing it on ice.
  3. Then we prepared the master mix using the following measurements to make 130% excess of master mix 1:
    Master Mix Rxn: 1 Rxns: 11
    CutSmart buffer 10x .9 uL 9.9 uL
    EcoRI-HF enzyme .28 uL 3.08 uL
    MSPI enzyme .12 uL 1.32 uL
    Pure H2O 1.7 uL 18.7 uL
    Master Mix Total: 3 ul 33 ul
  4. We mixed it well, centrifuged it and stored it on ice.
  5. Then, we added 3uL of MM1 to each DNA sample
  6. We sealed the samples, vortexed, centrifuged, and incubated them at 37 degrees Celsius for 8 hours.

My samples were labeled #3 (SCHO002-26) and #4 (DIRA006-11).

Then, we did an adapter ligation. We did this using the following steps:

  1. First, we thawed the working stock EcoRI and Mspl adapters previously made as follows:
    PAUL LAB ID Adapter name
    Eco_2 AACCA_EcoRI
    Eco_3 CGATC_ EcoRI
    Eco_4 TCGAT_ EcoRI
    Eco_5 TGCAT_ EcoRI
    Eco_6 CAACC_ EcoRI
    Eco_7 GGTTG_ EcoRI
    Eco_8 AAGGA_ EcoRI
    Eco_9 AGCTA_ EcoRI
    Eco_10 ACACA_ EcoRI
  2. We added 1 uL of the working stock EcoRI adapter directly to the digested DNA.
  3. Then, we made a second master mix, making 130% excess with the following measurements:
Master Mix Rxn: 1 Rxns: 11
CutSmart buffer 10x .4 uL 4.4 uL
ATP (10mM) 1.3 uL 14.3 uL
T4 Ligase .2 uL 2.2 uL
Pure H2O .1 uL 1.1 uL
Universal P2 Mspl adapter 1.0 uL 11.0
Master Mix Total: 3 ul 33 ul

4. We added 3 uL of MM2 to the digested DNA

5. Finally, we sealed, vortexes, centrifuged, and incubated this at 16 degrees Celsius for 6 hours.